CN117587168B - LAMP-CRISPR/Cas12a visualized kit and method for identifying RHDV type 1 and RHDV type 2 - Google Patents
LAMP-CRISPR/Cas12a visualized kit and method for identifying RHDV type 1 and RHDV type 2 Download PDFInfo
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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Abstract
The invention provides a kit and a method for visually identifying RHDV type 1 and RHDV type 2 by LAMP-CRISPR/Cas12a, belonging to the technical field of pathogenic microorganism detection. The invention establishes the LAMP-CRISPR/Cas12a rapid detection kit and the method aiming at RHDV1 and RHDV2, the platform has higher sensitivity, can detect 10 copies/mu L of nucleic acid sample, has good specificity, can specifically identify RHDV1 and RHDV2 type strains, and has no cross reaction with other common pathogens of rabbits. Further using a lateral flow chromatography test strip and visual fluorescence to obtain macroscopic results within 1.5 h. The kit is a portable platform, has the advantages of rapidness, high sensitivity, high specificity, visualization and low equipment requirement, and can be used for clinic in remote rural areas and resource-restricted areas.
Description
Technical Field
The invention belongs to the technical field of pathogenic microorganism detection, and particularly relates to a kit and a method for visually identifying RHDV type 1 and RHDV type 2 by LAMP-CRISPR/Cas12 a.
Background
Rabbit hemorrhagic disease is an acute, virulent, highly contagious infectious disease caused by Rabbit Hemorrhagic Disease Virus (RHDV), which has extremely high mortality rate and seriously jeopardizes the development of rabbit farming. Compared with the classical RHDV called rabbit hemorrhagic disease virus type 1 (RHDV 1), the strain rabbit hemorrhagic disease virus type 2 (RHDV 2) has wider host range, multiple transmission ways, strong resistance in natural environment, great economic loss to the breeding industry, and the diagnosis tool which is efficient, accurate and can identify the two is particularly important for preventing and controlling the rabbit hemorrhagic disease epidemic situation.
Currently, detection methods such as PCR, fluorescent quantitative PCR, hemagglutination experiments, ELISA and the like are available. Immunological detection methods such as hemagglutination experiments and ELISA experiments have the defects of poor detection accuracy, difficult detection in a window period and the like, and etiology detection methods such as RT-PCR and fluorescent quantitative PCR require professional instruments, are generally laboratory detection, cannot achieve on-site rapid detection, and cannot meet the detection requirements of basic farms. At present, research on rapid visual identification of rabbit hemorrhagic disease viruses based on LAMP and CRISPR-Cas12a has no relevant report, and the method is applied to rapid identification of rabbit hemorrhagic disease virus type 1 and rabbit hemorrhagic disease virus type 2.
Disclosure of Invention
The invention provides a kit and a method for visually identifying RHDV1 type and RHDV2 type by LAMP-CRISPR/Cas12a, which can accurately identify RHDV1 and RHDV2 viruses and have the advantages of rapidness, high sensitivity, high specificity, visualization and low equipment requirement.
In order to solve the technical problems, the invention provides the following technical scheme:
the invention provides a kit for visually identifying RHDV type 1 and RHDV type 2 by LAMP-CRISPR/Cas12a, which comprises a LAMP amplification system and a CRISPR/Cas12a detection system; the LAMP amplification system comprises an outer primer pair F3 and B3 and an inner primer pair FIP and BIP; the nucleotide sequences of the outer primer pair F3 and the outer primer pair B3 in the LAMP amplification system aiming at RHDV1 are shown as SEQ ID No.13-14, and the nucleotide sequences of the inner primer pair FIP and BIP are shown as SEQ ID No. 15-16; the nucleotide sequences of the outer primer pair F3 and the outer primer pair B3 in the LAMP amplification system aiming at RHDV2 are shown as SEQ ID No.24-25, and the nucleotide sequences of the inner primer pair FIP and BIP are shown as SEQ ID No. 26-27; the CRISPR/Cas12a detection system comprises gRNA; the nucleotide sequence of the gRNA in the CRISPR/Cas12a detection system aiming at RHDV1 is shown as SEQ ID No.37; the nucleotide sequence of the gRNA in the CRISPR/Cas12a detection system aiming at RHDV2 is shown as SEQ ID No. 39.
Preferably, the LAMP amplification system further comprises loop primers LF and LB; the nucleotide sequence of the loop primer LB in the LAMP amplification system aiming at RHDV1 is shown as SEQ ID No. 17; the nucleotide sequences of loop primers LF and LB in the LAMP amplification system aiming at RHDV2 are shown in SEQ ID No. 28-29.
Preferably, the LAMP amplification system also comprises LAMP/RT-LAMP 2X premix, nucleic acid to be detected and enzyme-free water.
Preferably, the CRISPR/Cas12a detection system further comprises a LAMP amplification product, an rnase inhibitor, a NEBuffer 2.1, a Cas12a protein, a ssDNA reporter, and enzyme-free water.
Preferably, the kit further comprises a lateral flow chromatography test strip.
The invention provides application of the kit in preparation of products for identifying RHDV1 and RHDV 2.
The invention provides a method for visually identifying non-disease diagnosis purposes of RHDV1 and RHDV2 based on LAMP-CRISPR/Cas12a, which comprises the following steps: (1) extracting RNA of a sample to be detected; (2) Performing LAMP amplification in an LAMP amplification system by taking the RNA extracted in the step (1) as a template; (3) Enzyme-cutting the LAMP amplification product obtained in the step (2) in a CRISPR/Cas12a detection system and performing fluorescence detection; or the LAMP amplification product is subjected to enzyme digestion in a CRISPR/Cas12a detection system and is detected by a lateral flow chromatography test strip.
Preferably, the total volume of the LAMP amplification system is 25. Mu.L, 12.5. Mu.L of LAMP/RT-LAMP 2X premix, 2.5. Mu.L of primer mixture, 2. Mu.L of extracted RNA and 8. Mu.L of enzyme-free water; in the LAMP amplification system aiming at RHDV1, the final concentration of the outer primer pair F3 and B3 is 200nM respectively, the final concentration of the inner primer pair FIP and BIP is 1200nM respectively, and the final concentration of the loop primer LB is 600nM; in the LAMP amplification system aiming at RHDV2, the final concentration of the outer primer pair F3 and B3 is 200nM respectively, the final concentration of the inner primer pair FIP and BIP is 800nM respectively, and the final concentration of the loop primers LF and LB is 600nM respectively.
Preferably, the total volume of the CRISPR/Cas12a detection system is 20. Mu.L, 2. Mu.L of LAMP amplification product, 1. Mu.L of RNase inhibitor (final concentration 0.5U/. Mu.L), 2. Mu.L of gRNA (final concentration 500 nM), 2. Mu.L of NEBuffer 2.1, 1. Mu.L of Cas12a protein (final concentration 125 nM), 1. Mu.L of fluorescence quenching ssDNA reporter (final concentration 250 nM) and 11. Mu.L of enzyme-free water when performing fluorescence detection.
Preferably, the CRISPR/Cas12a assay total volume is 20. Mu.L, 2. Mu.L LAMP amplification product, 1. Mu.L RNase inhibitor (final concentration 0.5U/. Mu.L), 2. Mu.L gRNA (final concentration 500 nM), 2. Mu.L NEBuffer 2.1, 1. Mu.L Cas12a protein (final concentration 125 nM), 1. Mu.L biotin ssDNA reporter (final concentration 100 nM) and 11. Mu.L enzyme-free water when performing lateral flow chromatography dipstick assays.
Compared with the prior art, the invention has the following beneficial effects:
in order to rapidly, conveniently and specifically detect RHDV1 and RHDV2 viruses, the invention establishes a LAMP-CRISPR/Cas12a rapid detection method aiming at RHDV1 and RHDV2, the platform has higher sensitivity, can detect 10 copies/mu L of nucleic acid sample, has good specificity, can specifically identify RHDV1 and RHDV2 strains, and has no cross reaction with other common pathogens of rabbits.
The invention further utilizes the lateral flow chromatography test strip and the visual fluorescence to obtain macroscopic results within 1.5 h. In addition, 74 clinical samples were tested with the test systems for RHDV1 and RHDV2, respectively, with a rate of coincidence of 97.30% and 97.30% with fluorescent quantitative PCR detection, respectively, with higher sensitivity than fluorescent quantitative PCR. The kit and the constructed detection method have the advantages of rapidness, high sensitivity, high specificity, visualization and low equipment requirement, and can be used for clinic in remote rural areas and resource-restricted areas.
Drawings
FIG. 1 is a schematic diagram of the LAMP-CRISPR/Cas12a detection method combined with LFA (lateral flow chromatography test strip) and combined with a blue light meter.
FIG. 2 utilizes fluorescent LAMP to detect and screen three primer sets of RHDV1 and RHDV2, respectively; wherein A: RHDV1 primer Set 1, set 2, set 3, B: RHDV2 primer sets Set 1, set 2 and Set 3.
FIG. 3 shows the results of a cross-validation experiment for amplifying RHDV1 and RHDV2 using LAMP; wherein A: and (3) detecting a cross experimental result by using nucleic acid gel electrophoresis, and B: endpoint fluorescence values for crossover experiments with fluorescent LAMP, n=3, P <0.0001.
FIG. 4 shows the results of detection reaction temperature screening for RHDV1 and RHDV2, respectively, using fluorescent LAMP; wherein A: RHDV1 screening results, B: RHDV2 screening results.
FIG. 5 shows the results of screening the inner and outer primer ratios for RHDV1 and RHDV2 respectively by using fluorescent LAMP; wherein A: RHDV1 screening results, B: RHDV2 screening results.
FIG. 6 shows the results of detection loop primer concentration screening for RHDV1 and RHDV2, respectively, using fluorescent LAMP; wherein A: RHDV1 screening results, B: RHDV2 screening results.
Fig. 7 visualizes the detection results of LAMP on RHDV1 and RHDV2, respectively.
FIG. 8 LAMP combined CRISPR/Cas12a detection method results; wherein A: fluorescence value change curve graph of orthogonal experiment of screening of gRNA and optimizing Cas12a protein concentration in CRISPR stage of RHDV1 detection system, B: fluorescence value change curve graphs of orthogonal experiments of screening gRNA and optimizing Cas12a protein concentration in CRISPR stage of RHDV2 detection system; c: comparison of orthogonal experimental endpoint fluorescence values (three replicates); d: cross experiments of trans-cleavage activity verification of Cas12a protein and RHDV1/RHDV2 detection method in CRISPR stage; e: trans-cleavage activity validation and crossover experiments were performed under blue light.
FIG. 9 shows the results of a specificity evaluation of the LAMP-CRISPR/Cas12a detection method; wherein A and B: specific results of LAMP-CRISPR/Cas12a fluorescence detection platform were assessed by endpoint fluorescence values (n=3, ×p < 0.0001), C and D: specific results of LAMP-CRISPR/Cas12a fluorescent detection platform, E and F, were evaluated by imaging results under blue light: specific results for LAMP-CRISPR/Cas12a based LFA detection platform. A. C, E corresponds to the RHDV1 detection system and B, D, F corresponds to the RHDV2 detection system.
FIG. 10 sensitivity assessment results of LAMP-CRISPR/Cas12a detection method; wherein A and B: sensitivity results of LAMP-CRISPR/Cas12a fluorescence detection methods were evaluated by endpoint fluorescence values (n=3, ×p < 0.0001), C and D: sensitivity results of the LAMP-CRISPR/Cas12a fluorescence detection method, E and F, were evaluated by imaging results under blue light: sensitivity results of the LAMP-CRISPR/Cas12a based LFA detection method. A. C, E corresponds to the RHDV1 detection system and B, D, F corresponds to the RHDV2 detection system.
FIG. 11 evaluation of clinical practicality of the LAMP-CRISPR/Cas12a detection method; wherein A and B: the LAMP-CRISPR/Cas12a fluorescence detection method detects endpoint fluorescence values (threshold 55482, 57905, respectively) for 74 clinical samples, C and D: the LAMP-CRISPR/Cas12a fluorescence detection method detects the blue light irradiation results of 74 clinical samples, E and F: the results of 74 clinical samples were detected based on the LAMP-CRISPR/Cas12a LFA detection method; g and H: the qPCR method is used for detecting the results of 74 clinical samples, and the circled number indicates that the detection result of the sample is inconsistent with the method established in the experiment. A. C, E corresponds to the RHDV1 detection system and B, D, F corresponds to the RHDV2 detection system.
Detailed Description
The invention provides a kit for visually identifying RHDV type 1 and RHDV type 2 based on LAMP-CRISPR/Cas12a, which comprises a LAMP amplification system and a CRISPR/Cas12a detection system; the LAMP amplification system comprises an outer primer pair F3 and B3 and an inner primer pair FIP and BIP; the nucleotide sequences of the outer primer pair F3 and B3 aiming at RHDV1 are shown as SEQ ID No.13-14, and the nucleotide sequences of the inner primer pair FIP and BIP are shown as SEQ ID No. 15-16; the nucleotide sequences of the outer primer pair F3 and B3 aiming at RHDV2 are shown as SEQ ID No.24-25, and the nucleotide sequences of the inner primer pair FIP and BIP are shown as SEQ ID No. 26-27; the CRISPR/Cas12a detection system comprises gRNA; the nucleotide sequence of gRNA aiming at RHDV1 is shown as SEQ ID No.37; the nucleotide sequence of gRNA for RHDV2 is shown as SEQ ID No. 39. The LAMP amplification system of the kit also comprises loop primers LF or/and LB; the nucleotide sequence of the loop primer LB for RHDV1 is shown as SEQ ID No. 17; the nucleotide sequences of loop primers LF and LB for RHDV2 are shown in SEQ ID Nos. 28-29.
In the invention, a plurality of groups of primers of a conserved segment VP60 are respectively designed for RHDV1 (isolate WF2007, genBank accession number: FJ 794180) and RHDV2 (isolate SC2020/04, genBank accession number: MT 383749), then the primers are screened, the primer group with highest efficiency is selected, and no cross reaction between RHDV1 and RHDV2 systems is ensured. The loop primer is designed on the basis of an outer primer and an inner primer.
In the present invention, the total volume of the LAMP amplification system was 25. Mu.L, 12.5. Mu.L of LAMP/RT-LAMP 2X premix, 2.5. Mu.L of primer mix, 2. Mu.L of extracted RNA and 8. Mu.L of enzyme-free water. The primer mixture comprises outer primer pairs F3 and B3, inner primer pairs FIP and BIP, and loop primers LF and/or LB.
In the present invention, the configuration of the primer mixture for RHDV1 includes the steps of: diluting each primer to 100 mu M; adding 12 mu L of FIP/BIP and 2 mu L of F3/B3 and 6 mu L of LB, and adding enzyme-free water until the total volume is 100 mu L, thus obtaining the primer mixture of RHDV 1.
In the present invention, the configuration of the primer mixture for RHDV2 includes the steps of: diluting each primer to 100 mu M; adding FIP/BIP 8 μl each, F3/B3 2 μl each, LF/LB 6 μl each, and then
Adding enzyme-free water to the total volume of 100 mu L to obtain the primer mixture of RHDV 2.
In the LAMP amplification system aiming at RHDV1, the final concentration of the outer primer pair F3 and B3 is 200nM respectively, the final concentration of the inner primer pair FIP and BIP is 1200nM respectively, and the final concentration of the loop primer LB is 600nM; in the LAMP amplification system aiming at RHDV2, the final concentration of the outer primer pair F3 and B3 is 200nM respectively, the final concentration of the inner primer pair FIP and BIP is 800nM respectively, and the final concentration of the loop primers LF and LB is 600nM respectively.
In the invention, the CRISPR/Cas12a detection system further comprises LAMP amplification products, an RNase inhibitor, NEBuffer 2.1, cas12a protein and ssDNA reporter molecules. The LAMP amplification product is an amplified product of RNA extracted from a sample in the LAMP amplification system, and the using amount of the LAMP amplification product is 1-3 mu L. The concentration of the RNase inhibitor is 0.2-0.8U/. Mu.L, and the dosage is 0.5-1.5 mu.L. The concentration of the gRNA is 450-550 nM, and the dosage is 1.5-2.5 mu L. The NEBuffer 2.1 dosage is 1-3 mu L. The dosage of the Cas12a protein is 0.5-1.5 mu L, and the concentration is 50-250 nM. The dosage of the ssDNA reporter molecule is 0.5-1.5 mu L, and the concentration is 50-300 nM. The invention designs gRNA (which is composed of a spacer sequence and a repeated sequence, wherein the spacer sequence is 20-21 bases after PAM sequence) and ssDNA probe (5 'FAM-TTATT-BHQ1 3' or 5'6-FAM-TTATT-Biotin 3') aiming at fragments amplified by LAMP. The invention screens the gRNA and optimizes the protein concentration of Cas12a under the condition that fluorescence is generated by CRISPR/Cas12a cutting experiments.
In the present invention, the kit further comprises a lateral flow chromatography test strip. The lateral flow chromatography test strip is a test strip suitable for rabbit hemorrhagic disease virus detection. The test strip is preferably a CRISPR Cas12/13 Hybridetection test strip (purchased from Wobbe biological company (Nanjing, china), JY 0301), and a sample adding pad of the test strip is added with a colloidal gold-labeled mouse anti-FAM antibody, a C line-labeled streptavidin, and a T line-labeled anti-mouse IgG antibody. When the kit contains a test strip, the ssDNA probe is a Biotin ssDNA probe (5'6-FAM-TTATT-Biotin 3'). The principle of the invention utilizing test paper judging result is as follows: when the ssDNA reporter molecule is not cut, the streptavidin of the quality control line is combined with biotin on the ssDNA to capture the ssDNA reporter molecule, and the C line is developed; when ssDNA reporter is cut due to the activated trans-cleavage ability of Cas12a, the broken FAM ends continue to surge upwards, the anti-mouse IgG antibody on the detection line is combined with the colloidal gold-labeled mouse antibody bound on FAM, the FAM ends with colloidal gold are captured, and the T line is developed; only the C line is negative, and only the T line is positive.
The invention also provides a method for identifying the non-disease diagnosis purpose of RHDV1 and RHDV2 based on LAMP-CRISPR/Cas12a visualization, which comprises the following steps: (1) extracting RNA of a sample to be detected; (2) Performing LAMP amplification in an LAMP amplification system by taking the RNA extracted in the step (1) as a template; (3) And (3) performing enzyme digestion and fluorescence detection on the LAMP amplification product obtained in the step (2) in a CRISPR/Cas12a detection system. The LAMP-CRISPR/Cas12a detection method is combined with LFA to realize visualization, a portable detection platform is constructed, and the fluorescent visualization can be realized by carrying out blue light instrument detection on the CRISPR/Cas12a detection system after enzyme digestion is completed, and a schematic diagram is shown in figure 1. The invention sets RHDV type 1 and RHDV type 2 to be detected in two different systems, namely a RHDV type 1 detection system and a RHDV type 2 detection system.
In the present invention, the total volume of the LAMP amplification system was 25. Mu.L, 12.5. Mu.L of LAMP/RT-LAMP 2X premix, 2.5. Mu.L of primer mix, 2. Mu.L of extracted RNA and 8. Mu.L of enzyme-free water. The primer mixture comprises an outer primer pair, an inner primer pair and/or a loop primer. The invention aims at RHDV1, an outer primer pair: inner primer: the volume ratio of the loop primer pair is 1:6:1.5 or 1:6:3, a step of; for RHDV2, the outer primer pair: the volume ratio of the inner primer pair is 1:4:3. aiming at RHDV1, the final concentration of the outer primer pair F3 and B3 is 200nM respectively, the final concentration of the inner primer pair FIP and BIP is 1200nM respectively, and the final concentration of the loop primer LB is 600nM; for RHDV2, the final concentrations of the outer primer pair F3 and B3 were 200nM, the final concentrations of the inner primer pair FIP and BIP were 800nM, and the final concentrations of the loop primers LF and LB were 600nM, respectively.
In the invention, the LAMP amplification reaction conditions are 65-69 ℃ for 25-35 min, preferably 67 ℃ for 30min.
In the present invention, the total volume of the CRISPR/Cas12a detection system is 20. Mu.L, 2. Mu.L of LAMP amplification product, 1. Mu.L of RNase inhibitor (0.5U/. Mu.L), 2. Mu.L of gRNA (500 nM), 2. Mu.L of NEBuffer 2.1, 1. Mu.L of Cas12a protein (125 nM), 1. Mu.L of ssDNA reporter (250 nM fluorescence quenching probe/100 nM biotin probe).
In the invention, the reaction condition in the CRISPR/Cas12a detection system is that the reaction condition is that the reaction temperature is 35-38 ℃ and the reaction time is 25-35 min.
In the invention, the LAMP, namely loop-mediated isothermal amplification technology, is the most widely applied isothermal amplification technology at present, can finish amplification in a short time by using a water bath pot, has amplification efficiency which is 2-5 higher than that of common PCR, and can visualize detection results by matching with dyes; the CRISPR/Cas12a technology can be used for gene editing and pathogen detection, is a detection method which is most commonly coupled with an isothermal amplification technology to increase specificity, and after the Cas12a protein is combined with gRNA, the protein is further combined with a target sequence to activate cleavage activity, so that not only can target nucleic acid be specifically cleaved, but also a nearby ssDNA probe can be cleaved indiscriminately, if fluorescein and a quencher are added to two ends of ssDNA, fluorescent groups emit light after being cleaved, and can point to a positive result. The invention is related to a lateral chromatography test strip on the basis of the LAMP combined CRISPR/Cas12a technology, and more visual detection results are obtained.
The invention also provides application of the kit and the detection method in identifying RHDV1 and RHDV 2.
In the present invention, all components or reagents are commercially available as known to those skilled in the art unless otherwise specified.
The technical solutions of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
1. LAMP primer design
The LAMP primer comprises two outer primers F3 and B3, two inner primers FIP and BIP and possibly loop primers LF and LB. Multiple sets of primers for the conserved segment VP60 were designed for RHDV1 (isolate WF2007, genBank accession number: FJ 794180) and RHDV2 (isolate SC2020/04, genBank accession number: MT 383749), respectively, and primer Set 1 (Set 1), primer Set 2 (Set 2), and primer Set 3 (Set 3) were designed for RHDV1 and RHDV2, respectively, as shown in Table 1. Primer sequences were all synthesized from gold sri biotechnology company (south kyo, china).
TABLE 1 LAMP primer sequences
| Viral primers | Sequence(s) |
| RHDV1（Set 1）F3 | TCCCTGACATGTCATTCGTG（SEQ ID No.1） |
| RHDV1（Set 1）B3 | GGTTTGTGCCGGTTACCAC（SEQ ID No.2） |
| RHDV1（Set 1）FIP | CGGGGGCACCGTTGTTACTGTTAACAGCCCCAACATTCCG（SEQ ID No.3） |
| RHDV1（Set 1）BIP | AGTTAGGTTTTGCCACTGGGGCCAGTCTGTGCACCTGAAGTG（SEQ ID No.4） |
| RHDV1（Set 1）LF | ACCACCAAACCCGACCCAC（SEQ ID No.5） |
| RHDV1（Set 1）LB | AACAGCCTCCAGCCCACCA（SEQ ID No.6） |
| RHDV1（Set 2）F3 | GCATGCAGTTCCGCTTCA（SEQ ID No.7） |
| RHDV1（Set 2）B3 | ACTAGTGTGGGGACAAGGC（SEQ ID No.8） |
| RHDV1（Set 2）FIP | CTCCAACCCTGGCCCAATCTCGTGTGTTTGGTGGGCGAC（SEQ ID No.9） |
| RHDV1（Set 2）BIP | CGCCCGTTCACTCGAACCTGCAGGGTCACCAGTTGGATG（SEQ ID No.10） |
| RHDV1（Set 2）LF | CCTGGTGGTATCACAGCCGC（SEQ ID No.11） |
| RHDV1（Set 2）LB | TCACCATGCCAGACTTGCGT（SEQ ID No.12） |
| RHDV1（Set 3）F3 | AACGGCAGCACATATGGC（SEQ ID No.13） |
| RHDV1（Set 3）B3 | GCTGTTAAAGGGCACGAATG（SEQ ID No.14） |
| RHDV1（Set 3）FIP | ACGTTGGTGGAGTTGTTCCCAGTTTGCCGACATTGACCATCG（SEQ ID No.15） |
| RHDV1（Set 3）BIP | GGTACGCTAATGCTGGGTCTGCATGTCAGGGAAGCCGTCT（SEQ ID No.16） |
| RHDV1（Set 3）LB | GATTGACAACCCTATCTCCCAGGTT（SEQ ID No.17） |
| RHDV2（Set 1）F3 | ACCACCGGGCATTGAGAT（SEQ ID No.18） |
| RHDV2（Set 1）B3 | TCGTGGATCCACCAAATGG（SEQ ID No.19） |
| RHDV2（Set 1）FIP | TGACTGGTTCGAGTGAACGAGCTGGGCCAGGTTTGGAAGT（SEQ ID No.20） |
| RHDV2（Set 1）BIP | GCGCCCCAACATGTACCACCAACGCTCAGGACCAACGT（SEQ ID No.21） |
| RHDV2（Set 1）LF | TGACAACATGAGGGAATTGTCTG（SEQ ID No.22） |
| RHDV2（Set 1）LB | CAACAGGCAACCCTGGC（SEQ ID No.23） |
| RHDV2（Set 2）F3 | ATGCTAGTGCCGGGTCTG（SEQ ID No.24） |
| RHDV2（Set 2）B3 | GTTGCTCGGTACTCCAGTG（SEQ ID No.25） |
| RHDV2（Set 2）FIP | GGTAGGGATGGTGATACCGCTGAACCCCATCTCCCAAATTGC（SEQ ID No.26） |
| RHDV2（Set 2）BIP | GGTCGGGTTCGGTGGGATCTTAAGCCTGCATGGTCGTGA（SEQ ID No.27） |
| RHDV2（Set 2）LF | TGACATGTCAGGGAAACCATCTGG（SEQ ID No.28） |
| RHDV2（Set 2）LB | AACAGCAGTAATGGTGCCCCC（SEQ ID No.29） |
| RHDV2（Set 3）F3 | CATGTACCACCCAACAGGC（SEQ ID No.30） |
| RHDV2（Set 3）B3 | GTGAGAACTGGGGTTGTGAG（SEQ ID No.31） |
| RHDV2（Set 3）FIP | CTCGTGGATCCACCAAATGGGTGTTCCCACGTTGGTCCTG（SEQ ID No.32） |
| RHDV2（Set 3）BIP | AGTTTGTGATGATCCGTGCCCCGAGATCTGCGGGCGAGAT（SEQ ID No.33） |
| RHDV2（Set 3）LF | TGATGAGGTTGTTGTAAACGCT（SEQ ID No.34） |
| RHDV2（Set 3）LB | TCCAGTAAGACCGTTGACTCG（SEQ ID No.35） |
2. Extraction of RNA templates
RNA of RHDV1 and RHDV2 strains (stored in the laboratory) were extracted respectively using an RNA extraction kit (purchased from Noruzan biosystems (Nanjing, china)) at a concentration of 218.6 ng/. Mu.L for RHDV1 strains and at a concentration of 158.8 ng/. Mu.L for RHDV2 strains, and used in the subsequent experiments.
3. LAMP amplification method is established aiming at RHDV1 and RHDV2 and primer and reaction conditions are optimized
(1) LAMP amplification step: and (3) taking the RNA extracted in the step (2) as a template, adding samples by using designed primers according to a reaction system in the table (2), uniformly mixing, and reacting for 30min at the temperature of 67 ℃ in a constant-temperature oscillating box. Wherein the configuration of the primer mixture for RHDV1 comprises the steps of: diluting each primer to 100 mu M; adding 12 mu L of FIP/BIP, 2 mu L of F3/B3, 6 mu L of LB/LF or 6 mu L of LB, and adding enzyme-free water until the total volume is 100 mu L to obtain the primer mixture of RHDV 1. The configuration of the primer mixture for RHDV2 comprises the following steps: diluting each primer to 100 mu M; adding 8 mu L of FIP/BIP, 2 mu L of F3/B3 and 6 mu L of LF/LB, and adding enzyme-free water until the total volume is 100 mu L to obtain the primer mixture of RHDV 2. LAMP amplification systems for RHDV1 and RHDV2, and the volume and final concentration of each primer are shown in tables 2-4.
TABLE 2 LAMP amplification System
| System component | Volume of | Initial concentration |
| LAMP/RT-LAMP 2X premix | 12.5μL | / |
| Primer mixture | 2.5μL | / |
| Extracted RNA | 2μL | RHDV1 218.6 ng/. Mu.L or RHDV 2.8 ng/. Mu.L |
| Enzyme-free water | 8μL | / |
TABLE 3 volumes and final concentrations of each primer in LAMP amplification System for RHDV1
| Primer(s) | Initial concentration | Volume of | Final concentration |
| FIP | 100μM | 0.3μL | 1.2μM |
| BIP | 100μM | 0.3μL | 1.2μM |
| F3 | 100μM | 0.05μL | 0.2μM |
| B3 | 100μM | 0.05μL | 0.2μM |
| LB/LF or LB | 100. Mu.M/100. Mu.M or 100. Mu.M | 0.15. Mu.L/0.15. Mu.L or 0.15. Mu.L | 0.6. Mu.M/0.6. Mu.M or 0.6. Mu.M |
TABLE 4 volume and final concentration of each primer in LAMP amplification System for RHDV2
| Primer(s) | Initial concentration | Volume of | Final concentration |
| FIP | 100μM | 0.2μL | 0.8μM |
| BIP | 100μM | 0.2μL | 0.8μM |
| F3 | 100μM | 0.05μL | 0.2μM |
| B3 | 100μM | 0.05μL | 0.2μM |
| LF | 100μM | 0.15μL | 0.6μM |
| LB | 100μM | 0.15μL | 0.6μM |
(2) Primer screening: based on the primers designed in step 1 and the LAMP amplification step in step 3 (1), it is set that a nucleic acid dye SYTO9 (final concentration of 250 nM) is added into the LAMP system, and the primer set with highest efficiency is selected through the change of fluorescence value under the monitoring of the Quant Studio 1 system, and no cross reaction between the RHDV1 and RHDV2 systems is ensured. The experimental procedure was negative with water. The results are shown in FIG. 2.
As can be seen from FIG. 2, the primers with the highest amplification efficiency are primer set 3 of RHDV1 and primer set 2 of RHDV 2.
Cross experiments were performed using the selected primer set 3 for RHDV1 and primer set 2 for RHDV2 to amplify the nucleic acids of RHDV1 and RHDV2, respectively. Gel electrophoresis analysis and fluorescence value analysis are carried out on the amplified result. The results are shown in FIG. 3.
As can be seen from fig. 3, by using gel electrophoresis to examine the crossover experiment, when amplifying RHDV1 with the selected primer set 3 for RHDV1, a trapezoidal band conforming to the expectation can be obtained, when amplifying RHDV2, no band is used, when amplifying RHDV1 and RHDV2 with the selected primer set 2 for RHDV2, respectively, no band is used, when amplifying RHDV1, a trapezoidal band conforming to the expectation can be obtained when amplifying RHDV 2; the results of the cross-over experiments using fluorescent LAMP showed a significant increase in fluorescence value only when amplifying RHDV1 nucleic acid with the primer set for RHDV1 and amplifying RHDV2 nucleic acid with the primer set for RHDV 2. The above results indicate that the two primer sets selected have better specificity.
(3) Reaction temperature, inner and outer primer concentration ratio, and loop primer concentration
Based on the screened primer group, the amplification step and the LAMP amplification system, the optimal reaction temperature, the concentration ratio of the outer primer to the inner primer and the reaction condition of the loop primer concentration are selected according to the change trend of the fluorescence value. Wherein the reaction temperature for the screening is set as: 61 ℃, 63 ℃, 65 ℃, 67 ℃, 69 ℃; the concentration ratio of the outer primer to the inner primer for screening is set as follows: 200nM:400nM, 200nM:800nM, 200nM:1200nM, 200nM:2000nM; the concentrations of the loop primers screened were 200nM, 400nM, 600nM, 800nM, 1000nM, respectively. The experimental procedure was negative with water. The results are shown in FIGS. 4-7.
As can be seen from FIGS. 4-7, the optimal reaction conditions for RHDV1 were 67℃and the outer primer: inner primer = 1:6 (200 nM:1200 nM), loop primer concentration 600nM, optimal reaction conditions for RHDV2 at 67 ℃, outer primer: inner primer = 1:4 (200 nM:800 nM), loop primer concentration 600nM. After neutral red dye is added into the LAMP system, the visualization of the result can be realized, and obvious color difference exists between the yin and yang results.
4. Establishing a CRISPR/Cas12a detection method aiming at RHDV1 and RHDV2 and optimizing a reaction system
(1) gRNA and ssDNA probe design
The gRNAs are designed by target fragments of RHDV1 and RHDV2 amplified in the preferred LAMP stage, and two gRNAs (gRNA 1 and gRNA 2) are designed respectively (the interval sequence is composed of a PAM sequence and a repeated sequence, the interval sequence is 20-21 bases after the PAM sequence), and the ssDNA probe (5 'FAM-TTATT-BHQ1 3') is designed. Both the gRNA and ssDNA probe sequences were synthesized from kusnezoff biotechnology company (south kyo, china) and are shown in table 5, where U in the table sequence is T in the corresponding sequence listing.
TABLE 5 gRNA sequence
| Virus (virus) | PAM | Sequence(s) |
| gRNA1 of RHDV1 | TTTG | UAAUUUCUACUAAGUGUAGAUCCGACAUUGACCAUCGAAGA（SEQ ID No.36） |
| gRNA2 of RHDV1 | TTTG | UAAUUUCUACUAAGUGUAGAUGUACGCUAAUGCUGGGUCUG（SEQ ID No.37） |
| gRNA1 of RHDV2 | TTTG | UAAUUUCUACUAAGUGUAGAUUACCCUUCAGCGGUAUCACCA（SEQ ID No.38） |
| gRNA2 of RHDV2 | TTTC | UAAUUUCUACUAAGUGUAGAUCCUGACAUGUCAUUUGUACCC（SEQ ID No.39） |
(2) CRISPR/Cas12a detection step: and taking the fragments amplified by the selected optimal LAMP amplification system and amplification conditions as templates, adding samples according to the reaction system in the table 6, uniformly mixing, and incubating at 37 ℃ for 30min to obtain a reactant solution.
TABLE 6 CRISPR/Cas12a detection System
| System component | Volume of | Final concentration |
| LAMP amplification products | 2μL | / |
| RNase inhibitors | 1μL | 0.5U/μL |
| gRNA | 2μL | 500nM |
| NEBuffer 2.1 | 2μL | / |
| Cas12a proteins | 1μL | 125nM |
| ssDNA reporter molecules | 1μL | 250nM |
| Enzyme-free water | 11μL | / |
(3) Screening of gRNA and optimization of Cas12a protein concentration by CRISPR/Cas12a cleavage assay to generate fluorescence: based on the primer gRNA in step 4 (1) and the step 4 (2) detection step, the change in fluorescence value was recorded using quantsudio 1 and blue fluorescent lamp irradiation, the result was judged by fluorescence profile and whether fluorescence was generated, and no cross-reaction between RHDV1 and RHDV2 systems was ensured. The fluorescence generated was verified to be generated by the gRNA-guided Cas12a protein binding to the target fragment activating its trans-cleavage activity, non-specifically cleaving surrounding ssDNA. In the experimental process, water is used as a negative control, and the amplification product with enzyme-free water as a template in the LAMP stage is LAMP-NC.
The grnas 1 and 2 of RHDV1 and RHDV2 were optimally screened, respectively, while orthogonal experiments were performed with 3 Cas12a protein concentrations (50 nM, 125nM, 250 nM) set, and the results are shown in fig. 8. As can be seen from fig. 8, at three different Cas12a concentrations, gRNA2 showed higher efficiency of cleavage fluorescence in each of the grnas designed for RHDV1, and gRNA2 showed higher efficiency of cleavage fluorescence in each of the grnas designed for RHDV 2; when the Cas12a concentration was increased from 50nM to 125nM, the fluorescence value generated was significantly increased (P < 0.05), and when the Cas12a concentration was further increased to 250nM, the difference in fluorescence value generated was not significant (P > 0.05) compared to 125nM, so the reaction concentration of Cas12a protein was set to 125nM.
Cas12a protein trans-cleavage activity validation and crossover experiments between RHDV1 and RHDV2 reaction systems were performed with selected grnas. And respectively using the products amplified by the selected optimal LAMP amplification system and the amplification conditions as templates of CRISPR/Cas12a cutting experiments of RHDV1 and RHDV2 systems, using enzyme-free water as a template as a negative control, and using a Quantum studio 1 system to detect the generated fluorescence. The results are shown in FIG. 8. The results show that there is a significant increase in fluorescence value in the system only when the template is the product of amplification of RHDV1 nucleic acid by RHDV1 primer and amplification of RHDV2 nucleic acid by RHDV2 primer, as well as the same result observed under blue light, which indicates that fluorescence generated by RHDV1 and RHDV2 systems during the CRISPR/Cas12a cleavage assay phase is generated by trans cleavage activity stimulated by binding of Cas12a protein to the target fragment under the guidance of the corresponding gRNA, and that there is no cross-reaction between RHDV1 and RHDV2 systems during this phase.
Example 2 LAMP-CRISPR/Cas12 a-based visual identification of RHDV type 1 and type 2 kit 1
The kit comprises a LAMP amplification system and a CRISPR/Cas12a detection system.
The total volume of the LAMP amplification system was 25. Mu.L, containing 12.5. Mu.L of LAMP/RT-LAMP 2X premix, 2.5. Mu.L of primer mix, 2. Mu.L of extracted RNA and 8. Mu.L of enzyme-free water. In the LAMP amplification system aiming at RHDV1, the primer group is shown as RHDV1 primer group 3 in table 1; the final concentration of the outer primer pair F3 and B3 is 200nM respectively, the final concentration of the inner primer pair FIP and BIP is 1200nM respectively, and the final concentration of the loop primer LB is 600nM; in the LAMP amplification system aiming at RHDV2, the primer group is shown as RHDV1 primer group 2 in table 1; the final concentration of the outer primer pair F3 and B3 is 200nM, the final concentration of the inner primer pair FIP and BIP is 800nM, and the final concentration of the loop primer pair LF and LB is 600nM.
The total volume of CRISPR/Cas12a detection system was 20. Mu.L, containing 2. Mu.L of LAMP amplification product, 1. Mu.L of RNase inhibitor (final concentration 0.5U/. Mu.L), 2. Mu.L of gRNA (final concentration 500 nM), 2. Mu.L of NEBuffer 2.1, 1. Mu.L of Cas12a protein (final concentration 125 nM), 1. Mu.L of ssDNA reporter (final concentration 250nM fluorescence quenching probe/100 nM biotin probe). Wherein, for RHDV1, the gRNA is shown as RHDV1 gRNA2 in Table 5; for RHDV2, the gRNA used is shown in Table 5 for RHDV2 gRNA2.
Example 3 LAMP-CRISPR/Cas12 a-based visual identification of RHDV type 1 and type 2 kit 2
The kit contained a lateral flow chromatographic test strip in addition to the system described in example 2. The lateral flow chromatography test strip is CRISPR Cas12/13 Hybridetect test strip (purchased from Wobbe biological company (Nanjing, china), JY 0301). The LAMP amplification system and CRISPR/Cas12a detection system were the same as in example 2, with the fluorescence quenching ssDNA probe replaced with a Biotin ssDNA probe (5'6-FAM-TTATT-Biotin 3').
Example 4
A method for identifying RHDV type 1 and type 2 based on LAMP-CRISPR/Cas12a visualization, comprising the steps of:
(1) Extracting an RNA solution of a sample to be detected with an RNA extraction kit (purchased from Northenan Bio Inc. (Nanjing, china));
(2) Adding the extracted RNA solution into the LAMP amplification system described in the embodiment 2 or 3, uniformly mixing, and reacting for 30min at 67 ℃ in a constant-temperature oscillating box to obtain an LAMP amplification product;
(3) Adding the obtained LAMP amplification product into the CRISPR/Cas12a detection system described in the embodiment 2 or 3, uniformly mixing, and incubating at 37 ℃ for 30min for enzyme digestion reaction to obtain a reactant solution after enzyme digestion;
(4) Irradiating the reactant solution with blue light instrument (LABGIC LANJIECO, beijing, china), and indicating positive when the reactant solution has fluorescence, and indicating negative when the reactant solution has no fluorescence;
the lateral flow test strip described in example 3 may also be placed in a reagent solution as follows, positive only when the T line is developed and negative only when the C line is developed. The operation steps comprise: 5. Mu.L of the reaction solution was taken, 45. Mu.L of enzyme-free water was added thereto, and a lateral flow chromatography test strip was inserted for color development.
Example 5 methodological evaluation of established LAMP-CRISPR/Cas12a detection method
(1) Specificity experiments: the specificity of the established detection method was verified by performing LAMP-CRISPR/Cas12a detection using the method described in example 4 to extract nucleic acids of 7 pathogens, RHDV1, RHDV2, E.coli (E.coli), salmonella typhi (S.tyrphi), klebsiella pneumoniae (K.pnumoniae), pasteurella multocida (P.multocida), rotavirus (RV). The experimental procedure was negative with water. The results are shown in FIG. 9. The result shows that when the RHDV1 detection method is used for detection, only RHDV1 is positive, and other pathogens are negative; when the detection method aiming at RHDV2 is used for detection, only RHDV2 is positive, and other pathogens are negative; the results seen in blue light are consistent with those shown by the test strips. This indicates that the detection methods established for both RHDV1 and RHDV2 are highly specific.
(2) Sensitivity experiment: cloning target genes by using RHDV1 isolate WF2007 and RHDV2 isolate SC2020/04 VP60 gene full-length primers, connecting amplified gene sequences to a pMD18-T vector, screening and sequencing to obtain positive pMD18-T-WF2007-VP60 and pMD18-T-SC2020-VP60 recombinant plasmids. The recombinant plasmid was subjected to concentration measurement using Nano drop and copy number was calculated using dd H 2 O dilution of recombinant plasmid to 1X 10 10 The copies/. Mu.L was used as template standard. Dilution with 10-fold ratio (1X 10) 6 Copy/. Mu.L-1X 10 0 Copy/. Mu.L) of VP60 full-length plasmids of RHDV1 and RHDV2 as detection templates were detected by the method described in example 4 (replacement of the extracted RNA solution with recombinant plasmid). The experimental procedure was negative with water. The results are shown in FIG. 10. The results indicate that the established LAMP-CRISPR/Cas12a detection method aiming at RHDV1 and RHDV2 can detect 1 multiplied by 10 at the lowest 1 Copy/. Mu.L of dsDNA template, the results seen in blue light are consistent with those shown by the test strips.
Example 6 clinical sample detection
74 clinical samples from different regions and farms were tested using qPCR method and LAMP-CRISPR/Cas12a detection method, and compliance and sensitivity of qPCR method and established LAMP-CRISPR/Cas12a detection method were compared. Water was used as a Negative Control (NC) at a concentration of 1X 10 5 The copies/. Mu.L standard positive plasmid (RHDV 1 or RHDV 2) was used as Positive Control (PC)
The LAMP-CRISPR/Cas12a detection method is the same as in example 4.
The qPCR detection step comprises the following steps: RNA extraction was performed on 74 clinical samples and qPCR amplification was performed with qPCR primers as required by the Vazyme FastPure Cell/Tissue Total RNA Isolation Kit V instructions. The absolute expression level of viral RNA in each tissue sample was measured in a Quant Studio 1 fluorescent quantitative PCR apparatus to determine the viral content in the different tissue samples. Triplicate assays were performed for each sample. The Ct value of a positive reference substance of the TaqMan probe real-time fluorescence quantitative PCR method is less than or equal to 35, and an amplification curve has obvious logarithmic growth phase; meanwhile, under the condition that the negative control amplification curve has no logarithmic growth phase, detecting that the Ct value of the sample is less than or equal to 35.0, and judging that RHDV1 or/and RHDV2 nucleic acid exists in the sample when a standard S-shaped amplification curve appears; and if the Ct value is not higher than 38 and the standard amplification curve is not available, judging that the RHDV1 or/and RHDV2 nucleic acid is negative. When the Ct value of the detection sample is less than or equal to 35.0 and less than or equal to 38, and the amplification curves are all standard S-shaped curves, the detection sample is judged to be suspicious, repeated experiments are needed, if the detection sample is still positive after repeated, otherwise, the detection sample is negative.
Wherein, the qPCR primer sequence is a primer RHDV-F1 upstream of the VP60 gene sequence: 5'-TGGARMTWGGYTTRAGTGTDGAYG-3' (SEQ ID No. 40); a downstream primer: RHDV-R1:5'-CAGACATAAGAAAARCCATTGGYTG-3' (SEQ ID No. 41); RHDV1 probe sequence: 5'-FAM-TGAYTGAACTCATTGAYGTACGCCC-BHQ1-3' (5 '-FAM-SEQ ID No.42-BHQ 1-3'); RHDV2 probe sequence: 5'-VIC-TGTCAGAMCTTGTTGACATCCGCC-BHQ2-3' (5 '-VIC-SEQ ID No.43-BHQ 2-3'); primers and probes were synthesized by the company Shanghai, inc. of Biotechnology. In the above sequences, R represents A or G, M represents A or C, W represents A or T, Y represents C or T, and D represents G or A or T.
The qPCR amplification reaction system is as follows: 2x One Step RT-PCR Buffer III 10 [ mu ] L, upstream and downstream primers (10 [ mu ] mol/L) of 0.6 [ mu ] L each, ROX dye II (50 x) of 0.4 [ mu ] L each, fluorescent probes (10 [ mu ] mol/L) of 0.8 [ mu ] L each, RNA products of 2 [ mu ] L, dd H 2 O4. Mu.L. The qPCR amplification reaction procedure was as follows: 42 ℃ for 5min, 95 ℃ for 10 s;95℃for 5s,60℃for 34s,40 cycles.
Fluorescence of the reactant solutions obtained by the two detection methods was monitored by quantsudio 1; the reactant solution was detected by blue light irradiation and a lateral chromatography test strip, and the result is shown in fig. 11. The result shows that when the RHDV1 detection system is used, 19 RHDV1 positive samples are detected by the method, 17 RHDV1 positive samples are detected by the qPCR method, and the coincidence rate of the two detection methods is 97.30%; when the RHDV2 detection system is used, 32 RHDV2 positive samples are detected by the method, 30 RHDV2 positive samples are detected by the qPCR method, and the coincidence rate of the two detection methods is 97.30%; the results of directly observing fluorescence under blue light by naked eyes of the two detection systems are consistent with the results of the lateral chromatography test strips, and the two read-out strategies are mutually verified. The coincidence rate with the qPCR method also verifies the stability and reliability of the detection method, and shows that the detection method constructed by the invention can be regarded as a new diagnosis method for detecting and identifying RHDV1 and RHDV2, and has feasibility in clinical practice.
All data of the present invention were statistically analyzed using GraphPad Prism 8.0, and single-factor analysis of variance (ANOVA) was used between the different groups. All experiments were repeated at least 3 times and data were expressed as mean ± standard deviation. When p-value <0.05, the difference is considered statistically significant.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. Kit for visually identifying RHDV type 1 and RHDV type 2 based on LAMP-CRISPR/Cas12a, which is characterized by comprising a LAMP amplification system and a CRISPR/Cas12a detection system;
the LAMP amplification system comprises an outer primer pair F3 and B3 and an inner primer pair FIP and BIP; the nucleotide sequences of the outer primer pair F3 and the outer primer pair B3 in the LAMP amplification system aiming at RHDV1 are shown as SEQ ID No.13-14, and the nucleotide sequences of the inner primer pair FIP and BIP are shown as SEQ ID No. 15-16; the nucleotide sequences of the outer primer pair F3 and the outer primer pair B3 in the LAMP amplification system aiming at RHDV2 are shown as SEQ ID No.24-25, and the nucleotide sequences of the inner primer pair FIP and BIP are shown as SEQ ID No. 26-27;
the CRISPR/Cas12a detection system comprises gRNA; the nucleotide sequence of the gRNA in the CRISPR/Cas12a detection system aiming at RHDV1 is shown as SEQ ID No.37; the nucleotide sequence of the gRNA in the CRISPR/Cas12a detection system aiming at RHDV2 is shown as SEQ ID No. 39.
2. The kit of claim 1, wherein the LAMP amplification system further comprises loop primers LF and/or LB; the nucleotide sequence of the loop primer LB in the LAMP amplification system aiming at RHDV1 is shown as SEQ ID No. 17; the nucleotide sequences of loop primers LF and LB in the LAMP amplification system aiming at RHDV2 are shown in SEQ ID No. 28-29.
3. The kit of claim 1, wherein the LAMP amplification system further comprises LAMP/RT-LAMP 2X premix, nucleic acid to be detected and enzyme-free water.
4. The kit of claim 1, wherein the CRISPR/Cas12a detection system further comprises LAMP amplification products, rnase inhibitors, NEBuffer 2.1, cas12a protein, ssDNA reporter molecules, and enzyme-free water.
5. The kit of claim 1, further comprising a lateral flow chromatographic test strip.
6. The use of a kit according to any one of claims 1 to 5 for the preparation of products for the identification of RHDV1 and RHDV 2.
7. A method for identifying RHDV type 1 and type 2 non-disease diagnostic purposes based on LAMP-CRISPR/Cas12a visualization, comprising the steps of:
(1) Extracting RNA of a sample to be detected;
(2) Performing LAMP amplification in an LAMP amplification system by taking the RNA extracted in the step (1) as a template;
(3) Enzyme-cutting the LAMP amplification product obtained in the step (2) in a CRISPR/Cas12a detection system and performing fluorescence detection;
or the LAMP amplification product obtained in the step (2) is subjected to enzyme digestion in a CRISPR/Cas12a detection system and is subjected to lateral flow chromatography test strip detection;
the LAMP amplification system comprises an outer primer pair F3 and B3 and an inner primer pair FIP and BIP; the nucleotide sequences of the outer primer pair F3 and the outer primer pair B3 in the LAMP amplification system aiming at RHDV1 are shown as SEQ ID No.13-14, and the nucleotide sequences of the inner primer pair FIP and BIP are shown as SEQ ID No. 15-16; the nucleotide sequences of the outer primer pair F3 and the outer primer pair B3 in the LAMP amplification system aiming at RHDV2 are shown as SEQ ID No.24-25, and the nucleotide sequences of the inner primer pair FIP and BIP are shown as SEQ ID No. 26-27;
the CRISPR/Cas12a detection system comprises gRNA; the nucleotide sequence of the gRNA in the CRISPR/Cas12a detection system aiming at RHDV1 is shown as SEQ ID No.37; the nucleotide sequence of the gRNA in the CRISPR/Cas12a detection system aiming at RHDV2 is shown as SEQ ID No. 39.
8. The method of claim 7, wherein the total volume of the LAMP amplification system is 25. Mu.L, 12.5. Mu.L of LAMP/RT-LAMP 2X premix, 2.5. Mu.L of primer mix, 2. Mu.L of extracted RNA, and 8. Mu.L of enzyme-free water;
in the LAMP amplification system aiming at RHDV1, the final concentration of the outer primer pair F3 and B3 is 200nM respectively, and the final concentration of the inner primer pair FIP and BIP is 1200nM respectively; in the LAMP amplification system aiming at RHDV2, the final concentration of the outer primer pair F3 and B3 is 200nM respectively, and the final concentration of the inner primer pair FIP and BIP is 800nM respectively.
9. The method of claim 7, wherein when performing fluorescent detection, the CRISPR/Cas12a detection system has a total volume of 20 μl,2 μl of LAMP amplification product, 1 μl of rnase inhibitor at a final concentration of 0.5U/μl,2 μl of gRNA at a final concentration of 500nM, 2 μl of NEBuffer 2.1, 1 μl of Cas12a protein at a final concentration of 125nM, 1 μl of fluorescence quenching ssDNA reporter at a final concentration of 250nM, and 11 μl of enzyme-free water.
10. The method of claim 7, wherein the total volume of the CRISPR/Cas12a assay system is 20 μl,2 μl of LAMP amplification product, 1 μl of rnase inhibitor at a final concentration of 0.5U/μl,2 μl of gRNA at a final concentration of 500nM, 2 μl of NEBuffer 2.1, 1 μl of Cas12a protein at a final concentration of 125nM, 1 μl of biotin ssDNA reporter at a final concentration of 100nM, and 11 μl of enzyme free water when subjected to lateral flow chromatography dipstick detection.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104046701A (en) * | 2014-06-12 | 2014-09-17 | 四川农业大学 | RT-LAMP (reverse transcription loop-mediated isothermal amplification) detection method for rabbit hemorrhagic disease virus (RHDV) |
| CN105277696A (en) * | 2015-10-26 | 2016-01-27 | 中国农业科学院上海兽医研究所 | Double-antibody sandwich ELISA (enzyme-linked immuno sorbent assay) detection kit for RHDV (rabbit hemorrhagic disease virus) and application method |
| CN112725539A (en) * | 2021-02-24 | 2021-04-30 | 遵义医科大学珠海校区 | PRA/Cas12a/IF kit of respiratory syncytial virus and detection method thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| EP3846837A4 (en) * | 2018-09-07 | 2022-09-14 | The J. David Gladstone Institutes, A Testamentary Trust Established under The Will of J. David Gladstone | Hiv or hcv detection with crispr-cas13a |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104046701A (en) * | 2014-06-12 | 2014-09-17 | 四川农业大学 | RT-LAMP (reverse transcription loop-mediated isothermal amplification) detection method for rabbit hemorrhagic disease virus (RHDV) |
| CN105277696A (en) * | 2015-10-26 | 2016-01-27 | 中国农业科学院上海兽医研究所 | Double-antibody sandwich ELISA (enzyme-linked immuno sorbent assay) detection kit for RHDV (rabbit hemorrhagic disease virus) and application method |
| CN112725539A (en) * | 2021-02-24 | 2021-04-30 | 遵义医科大学珠海校区 | PRA/Cas12a/IF kit of respiratory syncytial virus and detection method thereof |
Non-Patent Citations (2)
| Title |
|---|
| 兔出血症病毒可视化RT-LAMP检测方法的建立与应用;刘星星;耿毅;孙娜;陈勤;杨泽晓;李亚军;牟维豪;;中国兽医科学;20170620(第06期);全文 * |
| 环介导等温扩增技术在兔出血症病毒检测中的应用;原冬伟;刘家森;郭东春;姜骞;林欢;司昌德;曲连东;;中国兽医科学;20130420(第04期);全文 * |
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